The ability of various Interleukin-2 (IL-2) compounds to enhance an immune response has caused an enormous of commercial interest to focus on these compounds [see generally K. Welte and R. Mertelsmann, Cancer Investigation 3:35 (1985); M. H. Cheever et al., J. Biol. Resp. Modif. 3:462 (1984)]. The commercial aspects of this interest are reflected in the patent activity in this area: For example, U.S. Pat. No. 4,401,756 to Gillis discloses a process for preparing IL-2 from human malignant cells, U.S. Pat. No. 4,404,280 and 4,411,992 to Gillis disclose a process for producing murine (rat or mouse) IL-2 from malignant neoplastic cells, U.S. Pat. No. 4,448,879 to Fabricius and Stahn discloses a cell culture process for preparing serum-free and mitogen-free IL-2, U.S. Pat. No. 4,464,355 to Fabricius and Stahn discloses a serum-free and mitogen-free IL-2 derived from human, bovine, or porcine peripheral mononuclear blood cells, and U.S. Pat. No. 4,473,642 to Gillis discloses a process for producing murine IL-2 from hybridoma cells. These and other patents reflect the substantial importance of IL-2 compounds, and the importance of identifying additional IL-2 compounds to further expand the opportunities for their use.
IL-2, also referred to as T cell growth factor, is produced by activated T cells in response to antigenic stimulation, and is essential for the proliferation of activated T cells. IL-2 was first identified in the human system [D. A. Morgan et al., Science 193: 1007 (1976)], and has now been characterized in at least seven other mammalian species [L. S. English et al., Vet. Immunol. Immunopath. 9: 59 (1985)]. The size of the protein associated with naturally produced IL-2 varies, depending upon the animal species of origin. In human and rat, IL-2 is associated with a protein having a molecular weight of 15 kilodaltons (kda.) [S. Gillis et al., J. Immunol. 124: 1954 (1980); K. A. Smith et al., Mol. Immunol. 17: 579 (1980)], and in mouse with a protein having a molecular weight of 22 to 30 kda. [J. Watson et al., J. Exp. Med. 150: 849 (1979); D. Riendeau et al., J. Biol. Chem. 258: 12114 (1983)]. The cross species activity of IL-2 is variable, with human IL-2 having the broadest range in terms of its capacity to support the growth of activated T cells in other species, and with sheep IL-2 having the narrowest capability, supporting T cell growth only within its own species [P. Lindsay et al., in Human Lymphokines. The Biological Immune Response Modifiers, 479 (A. Kahn and N. Hill, eds., 1982); L. S. English et al., supra].
Several groups have begun to investigate avian IL-2, derived from activated avian T cells. K. Schauenstein et al. [Dev. Comp. Immunol. 6: 533 (1982)] were the first to present evidence for the presence of a T cell growth factor (an IL-2) in the supernatant of mitogen stimulated chicken spleen cells. While Schauenstein et al. did not purify this factor, it was observed that the chicken factor did not cross react with prestimulated murine lymphocytes, and that potent mouse T cell growth factor preparations did not exhibit a proliferative effect on chicken cells. After this development, Schauenstein's group disclosed an improved avian IL-2 production method [G. Kromer et al., J. Immunol. Methods 73: 273 (1984)].
Avian IL-2 activity has subsequently been suggested as associated with a 9-12 kda. protein by Schnetzler et al. [Eur. J. Immunol. 13: 560 (1983)], a 13-kda. protein by Vainio et al. [Scand. J. Immunol. 23: 135 (1986)], and a 19.5- to 21.5-kda. protein by Schnetzler et al., supra. The latter protein could be reduced to a 13-kda. molecule by SDS-polyacrylamide gel electrophoresis. It should be noted that the purified 13-kda. molecule described by Schnetzler et al. was not tested for IL-2 activity, and that Vainio et al. only show that IL-2 activity hovers around the 13-kda. region during Sephadex chromatography (see FIG. 3 therein). We now disclose the purification to homogeneity of a heretofor unknown species of avian IL-2 with a molecular weight of about 30 kda.